Fusogenic liposome-coated porous silicon nanoparticles

ABSTRACT

The disclosure describes a fusogenic liposome-coated porous silicon nanoparticles for high loading efficiency of anionic payloads (small molecules, dyes, nucleic acids), and for non-endocytic delivery of hydrophilic and lipophilic payloads by membrane fusion. The liposome coating can be further modified with targeting peptides or antibodies via covalent binding chemistry between the ligands and functionalized poly(ethylene glycol). The surface moieties can be transferred to the cellular membrane surface by fusogenic uptake. The composition of the disclosure can be applied in the treatment of diseases by delivering entrapped/encapsulated payloads.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Stage Application filed under 35U.S.C. § 371 and claims priority to International Application No.PCT/US2016/041639, filed Jul. 8, 2016, which application claims priorityunder 35 U.S.C. § 119 from Provisional Application Ser. No. 62/190,705,filed Jul. 9, 2015, the disclosures of which are incorporated herein byreference.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was funded by Grant No. HR0011-13-2-0017 awarded by theDefense Advanced Research Projects Agency (DARPA) and Grant No.DMR-1210417 awarded by the National Science Foundation. The governmenthas certain rights in the invention.

TECHNICAL FIELD

The invention relates to delivery systems and, more particularly,fusogenic liposomal nanoparticle compositions for delivery of drugs,nucleic acids and peptides to a target cell or tissue.

BACKGROUND

In vivo gene delivery remains a challenge due to low efficiency orcytotoxicity. Cellular endocytosis is the primary uptake pathway of mostnanoplatforms, which results in lysosomal degradation of geneticmaterial and low therapeutic efficacy.

SUMMARY

The disclosure provides a biodegradable liposomal porous siliconnanoparticle system that can bypass endocytic uptake via liposome-plasmamembrane fusion. Unlike most currently studied nanoparticular deliverysystems that allow for endocytic uptake of all its payloads, membranefusion allows direct release of hydrophilic payloads from the core ofliposomes into the cell cytoplasm, transfer of hydrophobic moleculesfrom the liposomal bilayer to the cell membrane bilayer, and transfer ofmoieties conjugated on the outer surface of liposomes (antibodies, smallmolecules, peptides, etc.) to the cell membrane surface.

Disclosed are liposomal porous silicon particles and their method ofsynthesis and applications. The liposome may be loaded with lipophilicpayloads, and be functionalized with poly(ethylene glycol) (PEG) andother surface moieties (targeting peptides, antibodies, aptamers, etc.)to transfer them into and on cellular membranes through fusion. A poroussilicon-based core can entrap high amounts of payloads (small molecule,protein, nucleic acid), and deliver them directly to the target cell'scytoplasm; by-passing cellular endocytosis can increase delivery andtherapeutic efficacy of treatment. In vitro knockdown efficiencycomparable to that of Lipofectamine has been demonstrated.

The disclosure provides a fusogenic liposome-coated porous siliconnanoparticle comprising, a nanostructured silicon-containing corematerial having a plurality of pores, the material comprising, (a) asilicon-containing core, (b) a porous surface that is chemically linkedto the core, (c) a plurality of cargo molecules that are physicallyassociated with silicon-containing core material, and (d) a metalsilicate; and a fusogenic liposome coating around the silicon-containingcore material. In one embodiment, the fusogenic liposome comprises1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG), and 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP). In another embodiment, the silicon-containing core issubstantially or completely oxidized silicon. In yet another embodimentof any of the foregoing embodiments, the metal silicate comprises acalcium or magnesium silicate. In a further embodiment, the metalsilicate forms a shell on the silicon-containing core. In yet anotherembodiment, there is no gaps between silicon-containing core and thecalcium or magnesium silicate shell. In another embodiment, theplurality of pores are chemically or physically configured to admit acargo molecule. In yet another embodiment, chemical oxidation of thepore surface in conjunction with the formation of a calcium or magnesiumsilicate shell results in physical trapping of the plurality of cargomolecules. In still another embodiment, the plurality of pores contain adrug. In another embodiment, the plurality of pores contain a non-drugsubstance. In another embodiment, the silicon-containing core comprisesa molecule that is physically, adsorbed, or covalently trapped by withthe plurality of pores, or attached to the plurality of pores, andwherein the silicon-containing core is coated by a calcium or magnesiumcontaining shell formed by the action of aqueous solution of calcium ormagnesium ions added to said host material in the presence of themolecule. In yet another embodiment, the fusogenic liposome comprises amixture of DMPC, DOTAP, and DSPE-PEG (methoxy). In another embodiment,the fusogenic liposome comprises a mixture of DMPC, DOTAP, and DSPE-PEG(carboxy). In another embodiment, the fusogenic liposome comprises amixture of DMPC, DOTAP, and DSPE-PEG (maleimide). In yet anotherembodiment, the silicon-containing core material has a hydrodynamicdiameter ranging from about 10-100 nm. In another embodiment, thefusogenic liposome has a hydrodynamic diameter ranging from about100-400 nm. In still yet another embodiment, a targeting molecule isconjugated to the fusogenic liposomal coating. In one embodiment, anantibody is conjugated to the fusogenic liposomal coating. In anotherembodiment, a hydrophobic cargo molecule is loaded in the fusogenicliposomal coating. In yet another embodiment, a hydrophilic cargomolecule is entrapped within the plurality of pores of thesilicon-containing core. In still another embodiment, a hydrophobicagent/cargo molecule is present in the fusogenic coating and ahydrophilic agent/cargo is present in the plurality of pores of thesilicon-containing core. In another embodiment, a nucleic acid cargomolecule is entrapped within the plurality of pores of thesilicon-containing core. In another embodiment, a small molecule cargomolecule is entrapped within the plurality of pores of the poroussilicon-containing core.

The disclosure also provides a method of delivering nucleic acidpayloads to cells comprising contacting the cell with a fusogenicliposome-coated porous silicon nanoparticle of the disclosure thatcontains a nucleic acid.

The disclosure also provides a method of treating a disease or disorderof the eye comprising delivering into or upon a surface of the eye thefusogenic liposome-coated porous silicon nanoparticle of the disclosure.

The disclosure also provides a method for treating cancer comprisingdelivering into the body the fusogenic liposome-coated porous siliconnanoparticle of the disclosure.

The disclosure also provides a method for treating bacterial infectioncomprising delivering into the body the fusogenic liposome-coated poroussilicon nanoparticle of the disclosure.

The details of one or more embodiments of the invention are set forth inthe accompanying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A-H show schematics representing embodiments of the disclosure.(A) shows a schematic of action of a fusogenic liposome-coated poroussilicon nanoparticle of the disclosure. (B) shows loading of calciumcoated porous silicon (pSi) with anionic payloads. Anionic payload andcationic metal-silicate-deposited pSiNPs form a cluster by electrostaticinteractions. (C) shows loading of “loaded”-pSi into cationic liposomes.The payload-pSi cluster are encapsulated into cationic fusogenicliposomes via electrostatic interactions and mechanical extrusion. (D)depicts modification of the liposome with targeting peptides. Targetingpeptide or antibody can be conjugated to functional PEG via chemicalbinding. (E) shows low magnification of particles. Imaged using TecnaiTEM. Negative staining done by uranyl acetate. Scale bar indicates 500nm. (F) shows high magnification of particles show cloudy liposomalcoating around dark and dense porous silicon-based core. Imaged usingJEOL 1200 EX TEM. Negative staining by 2% PTA. Scale bar indicates 200nm. (G) a table of particle size and zeta-potential measured by DLS(n=3). (H) shows a schematic of a particle of the disclosure.

FIG. 2 shows a schematic of drug loading of porous siliconnanoparticles.

FIG. 3 shows a schematic of cargo-loaded calcium silicate-coated poroussilicon nanoparticles (Ca-pSiNP).

FIG. 4A-I shows confocal microscopy and transmission electronmicroscopy. (A-F) Confocal microscopy of J771A.1; (A) J771A.1 after 10min incubation with F-pSi loaded with DiI; (B) J774a.1 after 1 hincubation with Lysotracker Red and 10 min incubation with F-pSi withcalcein; (C) J774a.1 after 5 min incubation with MTP-FAM conjugatedF-pSi loaded with DiI; (D) J771A.1 after 10 min incubation with NF-pSiloaded with DiI; (E) J774a.1 after 1 h incubation with Lysotracker Redand 10 min incubation with NF-pSi with calcein; (F) J774a.1 after 5 minincubation with MTP-FAM conjugated NF2-pSi loaded with DiI. (G-I)Transmission electron microscopy (TEM) of HeLa cells after 1 hincubation with particles; (G) Non-fusogenic liposome-coated particleslocalized in vesicles (endosome/lysosome). Inset shows pinocytoticuptake of particles. Scale bar indicates 1 μm; (H) Fusogenicliposome-coated particles localized in cell cytoplasm. Scale barindicates 500 nm; and (I) Fusogenic liposome-coated particles localizedin cell cytoplasm. Scale bar indicates 1 μm.

FIG. 5A-B shows confocal microscopy of Neuro2a cells after 1 hincubation with particles; (A) Fusogenic liposomes showed DiI signals inthe cell membrane, indicating fusogenic uptake; (B) Non-fusogenicliposomes showed DiI signal in the cell cytoplasm in distinctconcentrated spots, indicating endocytic uptake.

FIG. 6 shows confocal microscopy of HeLa cells up to 8 h of incubationwith fusogenic liposome-coated particles.

FIG. 7 shows confocal microscopy of HeLa cells up to 8 h of incubationwith non-fusogenic liposome-coated particles.

FIG. 8A-B shows (A) Mouse survival post-infection at day 0 andpost-therapeutics injection (PBS, NF-siIRF5-MTP, F-siLuc-MTP, andF-siIRF5-MTP) at day 1. Each group has n=6 mice. (B) Average days ofsurvival of mice post-infection at day 0 and post-therapeutic injectionat day 1. Error bar indicates standard deviation. One-way ANOVA withTukey's HSD post hoc test (α=0.05) revealed significant differencebetween F-siIRF5-MTP and the three control groups (PBS, NF-siIRF5-MTP,and F-siLuc-MTP).

FIG. 9 shows month-long observation of average hydrodynamic diameter offusogenic liposome-coated calcium silicate porous silicon nanoparticles(F-CapSi) and RVG-conjugated fusogenic liposome-coated calcium silicateporous silicon nanoparticles (RVG-F-CapSi) by DLS.

FIG. 10 shows absorbance (dotted) and emission (solid) spectra ofCalcein loaded in fusogenic liposome-coated calcium silicate poroussilicon nanoparticles (F-Ca-pSi) and non-coated calcium silicate poroussilicon nanoparticles (Ca-pSi).

FIG. 11 shows photoluminescence spectra obtained over reaction time ofpSiNPs and CaCl₂ (or MgCl₂) solution.

FIG. 12 shows confocal microscopy of Neuro2a and HeLa cells afterincubation with particles; Top left panel: Neuro2a cells after 1 hincubation with fusogenic liposome-coated particles loaded withlipophilic fluorescent dye, DiI; Top right panel: HeLa cells after 8 hincubation with fusogenic liposome-coated particles loaded with cellimpermeable dye, Calcein; Bottom left panel: Neuro2a cells after 1 hincubation with non-fusogenic liposome-coated particles loaded withlipophilic fluorescent dye, DiI; Bottom right panel: HeLa cells after 8h incubation with non-fusogenic liposome-coated particles loaded withcell impermeable dye, Calcein.

FIG. 13A-B shows Confocal microscopy of Neuro2a cells after 1 hincubation with particles; (A) RVG-conjugated fusogenic liposomes(RVG-F) show successful RVG targeting to cells and high level of fusionstaining; (B) Fusogenic liposomes (F) without RVG conjugation showfusion at a relatively lower level compared to RVG-conjugated particles.

FIG. 14 shows siRNA knockdown results from 48 h incubation of particlesin Neuro2a mouse neuroblastoma cells. F-Ca-pSi-siPPIB: Fusogenicliposome-coated calcium silicate porous silicon nanoparticles loadedwith siRNA against PPIB; NF-Ca-pSi-siPPIB: Non-fusogenic liposome-coatedcalcium silicate porous silicon nanoparticles loaded with siRNA againstPPIB; F-Ca-pSi-siLuc: Fusogenic liposome-coated calcium silicate poroussilicon nanoparticles loaded with siRNA against luciferase.

FIG. 15A-K shows FACS analyses of calcein accumulation in homogenizedStaph. aureus infected Balb/C lungs. (A) PBS vs. NF-pSi loaded withcalcein (Cal) and conjugated to targeting peptide (MTP); (B) PBS vs.F-pSi loaded with calcein without targeting peptide. (C) PBS vs. F-pSiloaded with calcein and conjugated to MTP. (D-K) unstained healthy orinfected lung histology sections imaged for fluorescence detection ofDiI-loaded particles.

DETAILED DESCRIPTION

As used herein and in the appended claims, the singular forms “a,”“and,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pore” includes aplurality of such pores and reference to “the antigen” includesreference to one or more antigens known to those skilled in the art, andso forth.

Also, the use of “or” means “and/or” unless stated otherwise. Similarly,“comprise,” “comprises,” “comprising” “include,” “includes,” and“including” are interchangeable and not intended to be limiting.

It is to be further understood that where descriptions of variousembodiments use the term “comprising,” those skilled in the art wouldunderstand that in some specific instances, an embodiment can bealternatively described using language “consisting essentially of” or“consisting of.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this disclosure belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice of the disclosed methods and compositions, the exemplarymethods, devices and materials are described herein.

The publications discussed above and throughout the text are providedsolely for their disclosure prior to the filing date of the presentapplication. Nothing herein is to be construed as an admission that theinventors are not entitled to antedate such disclosure by virtue ofprior disclosure.

Gene delivery systems for in vivo therapeutics remain a challenge due tolow efficiency or cytotoxicity. Cellular endocytosis is the primaryuptake pathway of most nanoplatforms, which results in lysosomaldegradation of genetic material and low therapeutic efficacy.

Porous silicon and silicon oxide have been investigated as candidatedrug delivery vehicle materials in many applications for their inorganicand biodegradable nature. The porous nanostructures can holdtherapeutics, diagnostic agents, or other beneficial substances(sometimes referred to herein as “payloads”). However, premature releaseof these payloads, either prior to or post-administration, can beundesirable for the intended purpose. Additionally, the degradation ofporous silicon or porous silicon oxide in aqueous conditions at sitesother than the target site has posed a significant problem for sustaineddrug delivery, in vivo or in vitro imaging, and biosensor applications,which can benefit from protective coating around the porous siliconcore.

Lipid-based nanoparticles were first Food and Drug Administration(FDA)-approved three decades ago in the form of Doxil®,doxorubicin-encapsulating liposome. Liposomal formulation of vincristinesulfate for acute lymphoblastic leukemia, Marquibo®, was approved asrecently as 2012, and several paclitaxel and cisplatin formulations suchas EndoTag-1 and SPI-077 are in clinical trials. The reason for suchcontinuous investment in liposomal formulations is attributed to theirmajor advantages: biocompatibility, ease of synthesis, and surfacemodifiability.

Lipid nanoparticles have difficulty in delivering genetic material dueto low loading efficiency and high rate of lysosomal degradation. Smallinterfering RNA (siRNA)-loaded lipid nanoparticles are taken up by cellsthrough clathrin-mediated endocytosis and macro/micropinocytosis withproton pump, mTOR, and cathepsin activations. After the uptake,approximately 70% of the internalized siRNA are exocytosed out of thecell after lipids comprising the nanoparticles are recycled at lateendosome/lysosome stages through Niemann-Pick type C1 (NPC1) regulation.Only 1-2% of the siRNA are able to escape from the early endosome intothe cytosol. Therefore, a delivery system that bypasses the lipidrecycling in late endosome/lysosome is vital in achieving hightherapeutic efficacy.

The preparation of nano-, micro-, meso-, and macroporous silicon byelectrochemical or chemical means is well established, as is theconversion of the material to the corresponding nano-, micro-, meso-,and macroporous silicon oxide, either partially or completely. Moreover,liposomes are known and recognized in the art

The disclosure provides a biodegradable liposomal porous silicon (pSi)nanoparticle system that can bypass endocytic uptake via liposome-plasmamembrane fusion (see, e.g., FIG. 1A). Membrane fusion allows a directrelease of hydrophilic payloads from the core of liposomes into the cellcytoplasm, as well as a transfer of hydrophobic molecules from theliposomal bilayer to the cell membrane bilayer. In addition, theliposomal porous silicon nanoparticle system allows a transfer ofmoieties conjugated on the outer surface of liposomes (antibodies, smallmolecules, peptides, etc.) to the cell membrane surface. The poroussilicon core has photoluminescence properties that allows for theseparticles to be used as a tracking tool using time-gated luminescenceimaging. Moreover, the porous silicon allows for condensation of thehighly anionic genetic material into small clusters, allowing liposomesto easily encapsulate the particle and any payloads contained in thepores of the particle. The disclosure demonstrates that fusogenicliposomal-coated pSi particles fuse with the membrane of cells andtransfer various payloads into the cell. For example, the disclosuredemonstrates that the fusogenic liposomal-coated pSi particlessuccessfully fuse with Neuro2a mouse neuroblast cells, and transferslipophilic DiI dye from the liposomal membrane to the cellular membrane.In contrast, non-fusogenic pSi particles were found in the cells insmall groups indicative of endosomal uptake and lysosomal localization.Additionally, surface conjugation of Neuro2a-targeting moiety, rabiesvirus glycoprotein (RVG), allowed an accelerated rate of fusion of theliposomal pSi. Overall, the liposomal porous silicon nanoparticlesdemonstrate potential as a highly effective delivery vehicle.

As used herein the term “microparticle” or “nanoparticle”, “micro-and/or nanoparticle”, “LPSiNP” and “pSiNP” refers to a porous siliconmaterial at least partially comprising silicon dioxide and which have asize range of a few nanometers to hundred micrometers. Typically thesize is about 10-20 nm to 1 micrometer. The geometry of the poroussilicon material/particles may be spherical, oblong, square,rectangular, cuboidal and the like.

It should be understood that here “porous silicon oxide” refers to asubstance containing silicon and oxygen of general stoichiometricformula SiOx, where x can be as small as 0.01 and as large as 2, andthat “porous silicon” refers to a substance that is composed ofelemental silicon (either in its crystalline or amorphous state), with asurface containing hydrogen, oxygen, or carbon-containing species. Herethe terms “porous silicon” or “porous silicon oxide” refer to materialscontaining micropores (pore sizes typically smaller than about 2 nm),mesopores (pore size typically in the range about 2-50 nm), ormacropores (pore sizes larger than about 50 nm), or combinations of anytwo or all three pore types. Further it should be understood that thesurface of the porous materials, including the surface of the inner porewalls, may contain hydrogen, oxygen, or carbon-containing species.

The disclosure provides porous silicon micro- and/or nanoparticles(pSiNP) that can carry one or more molecule to be delivered to a cell ortissue. The molecules can be diagnostic and/or therapeutic. In someembodiments, the molecules are anti-cancer agents, anti-inflammatoryagents, small molecule drugs, peptides, polypeptides, nucleic acids(e.g., siRNA) and the like.

Furthermore, in contrast to many micro- and nanomaterials (e.g., carbonnanotubes (CNT), gold nanoparticles (GN), and quantum dots (QD)), pSiNPdegrade into renally cleared components in a relatively short period oftime with little or no evidence of toxicity. Additionally, in contrastto many biologic-derived delivery systems, the nanoparticles alone(without an added activating complex or molecule) do not induce animmune response.

The porous silicon micro- and/or nano-particles containing a desiredpayload (e.g., nucleic acids, peptides, small molecules etc.) can beencapsulated into a liposome (fusogenic or non-fusogenic). The liposomecan be modified to be target specific or can be unmodified (see, e.g.,FIG. 1).

Accordingly, the disclosure provides a biodegradable porous micro-and/or nanostructure comprising silicon material encapsulated in aliposomal vesicle. In one embodiment, the silicon material comprises asilicon dioxide material. In another embodiment, the silicon materialcomprises both a silicon and a silicon dioxide material. In anotherembodiment, the biodegradable/biocompatible porous nanostructurecomprises a particulate size of between about 0.01 μm and 1 μm. In yetanother embodiment, the biodegradable/biocompatible porous structure canbe characterized as non-toxic. In yet another embodiment, the poroussilicon material is loaded with a “payload” material. The payloadmaterial can be a drug, small molecule, diagnostic agent, therapeuticagent, peptide, antibody, antibody fragment, polypeptide, nucleic acid(e.g., siRNA) and the like.

The disclosure also provides a method of preparing porous siliconparticles comprising (1) electrochemically etching a silicon wafer togenerate a porous structured film; (2) lifting off said porousstructured film from the silicon wafer substrate; (3) fracturing theporous film to generate micro- and/or nanoparticles of sizes between 10nanometers and 1000 nanometers; and (4) activating the structure in anaqueous solution. In one embodiment, the aqueous solution comprises purewater. In one embodiment, the aqueous solution comprises sodiumhydroxide, hydrogen peroxide or borate.

For example, porous silicon nanoparticles (pSiNPs) can be prepared asdescribe by Qin et al. (Part. Part. Syst. Charact. 31(2):252-256, 2014;the disclosure of which is incorporated herein by reference). Briefly,porous silicon is prepared by galvanostatic anodic etch of crystallinesilicon wafers. Perforations along the etched planes are introduced byshort periodic pulses of high current during a long low-current etch,generating layers of alternating high and low porosity. The layer ofporous silicon is removed from the wafer by applying low current densitypulse in dilute aqueous HF, and the resulting freestanding films arefractured by ultrasonication. This results in porous nanoparticles ofpredetermined porosity and average pore size. The tunability of porosityand pore size is useful in determining the efficiency of the subsequentpayload loading process.

In embodiments with payloads, the porous silicon particles can be placedin an aqueous solution containing the payload and 1M or greaterconcentration of, for example, calcium chloride (CaCl₂) solution. Thesolution is mixed and purified by centrifugation, resulting incargo-loaded calcium silicate coated porous silicon nanoparticle(Ca-pSiNP-cargo). As a control. Calcium silicate coated porous siliconnanoparticles without payload (Ca-pSiNP) can be prepared in the samemanner as described above, but excluding the added payload solution.FIGS. 1B, 2-3 illustrates the cargo-loaded calcium silicate-coatedporous silicon nanoparticle.

It will be apparent to those skilled in the art that other embodimentsmay be used to generate Ca-pSiNP, one example involving substituting achemical stain etch for the electrochemical etch used to produce theporous silicon core, and the other utilizing a porous silicon coreprepared by chemical reduction of a nanostructured silicon oxide. Stainetching uses silicon powder instead of silicon wafers as siliconprecursor, and a chemical oxidant instead of electrical power supply todrive the electrochemical reaction. In some cases, it may be desirableto substitute calcium nitrate, calcium nitrite, calcium gluconate, orother calcium ion sources for calcium chloride. Calcium nitrate orcalcium nitrite can oxidize porous silicon more quickly than calciumchloride due to the oxidizing nature of the nitrate and nitrite ions.

In one embodiment, the porous silicon particles can be luminescent. Thedisclosure provides a method of generating luminescent porous Sinanoparticles (LPSiNP). The method comprises electrochemical etching ofa p-type silicon wafer by application of a constant current density ofabout 200 mA/cm² in an aqueous HF/ethanol electrolyte. The resultingfreestanding film of porous silicon nanostructure is then removed fromthe crystalline silicon substrate by application of a current pulse ofabout 4 mA/cm² in an aqueous HF/ethanol electrolyte. The freestandinghydrogen-terminated porous silicon film is subsequently fractured, e.g.,by sonication, and then filtered to obtain a desired particle size.Other methods of size selecting the nanoparticles can be performed bycentrifugation and chromatography. The nanoparticles are furtherincubated in deionized (DI) water or other oxidizing aqueous environmentsuch as, for example, a borate aqueous buffer, to activate theirluminescence (e.g., in one embodiment in the near-infrared range).Various aqueous buffers that are oxidizing (or neutral to basic) can beused. In some embodiments, an aqueous buffer selected from the groupconsisting of an aqueous borate buffer, a phosphate buffered saline, andsodium hydroxide. For example, in one embodiment, a borate aqueousbuffer is useful. The resulting LPSiNP can then be further modified orloaded with a desired drug agent or other factor. The loaded or modifiedLPSiNP's can then be encapsulated by/into a liposome.

In another embodiment, the LPSiNP materials can be generated by firstproducing a silicon layer with a pore size range of 2-100 nm (e.g., 5-10nm, 10-20 nm, 20-30 nm etc.). The silicon layer is etched into thesingle-crystal silicon substrate in ethanolic HF solution. The entireporous nanostructure is removed from the Si substrate by application ofa current pulse. The freestanding hydrogen-terminated porous siliconfilm is then placed in an aqueous solution and fractured intomulti-sized particles by, for example, overnight ultrasonication. Theparticles can then be filtered if desired (e.g., through a 0.22 μmporous filtration membrane or other size separating device) to obtainporous silicon nanoparticles. For example, separation or size control ofLPSiNPs can be achieved by passing the colloidal suspension throughphysical filters, by centrifugation of the suspension, byelectrophoresis, by size exclusion chromatography, or by electrostaticprecipitation. The nanoparticles are incubated in an aqueous oxidizingsolution to activate their luminescence.

The activation of luminescence is performed in an aqueous solution.During the activation silicon oxide grows on the hydrogen-terminatedporous silicon surface, generating significant luminescence attributedto quantum confinement effects and to defects localized at the Si/SiO₂interface. The preparation conditions of the nanoparticles can beoptimized to provide pore volumes and surface areas suitable for loadingof therapeutics and for desired in vivo circulation times whilemaintaining an acceptable degradation rate.

The thickness, pore size, and porosity of a given film is controlled bythe current density, duration of the etch cycle, and etchant solutioncomposition. In addition, a porous silicon film can be used as atemplate to generate an imprint of biologically compatible orbioresorbable materials. The porous silicon film or its imprint possessa sinusoidally varying porosity gradient, providing sharp features inthe optical reflectivity spectrum that can be used to monitor thepresence or absence of chemicals trapped in the pores.

For in vivo applications, it is often desirable to prepare porous Si inthe form of particles. The porous layer can be removed from the Sisubstrate with a procedure commonly referred to as “electropolishing” or“lift-off.” The etching electrolyte is replaced with one containing alower concentration of HF and a current pulse is applied for severalseconds. The lower concentration of HF results in a diffusion limitedsituation that removes silicon from the crystalline Si/porous Siinterface faster than pores can propagate. The result is an undercuttingof the porous layer, releasing it from the Si substrate. Thefreestanding porous Si film can then be removed with tweezers or avigorous rinse. The film can then be converted into microparticles byultrasonic fracture. Conventional lithography or microdroplet patterningmethods can also be used if particles with more uniform shapes aredesired.

The ability to easily tune the pore sizes and volumes during theelectrochemical etch is a unique property of porous Si that is veryuseful for drug delivery applications. Other porous materials generallyrequire a more complicated design protocol to control pore size, andeven then, the available pore sizes tend to span a limited range. Withelectrochemically prepared porous Si, control over porosity and poresize is obtained by adjusting the current settings during etching.Typically, larger current density produces larger pores. Large pores aredesirable when incorporating sizable molecules or drugs within thepores. Pore size and porosity is important not only for drug loading; italso determines degradation rates of the porous Si host matrix.

Smaller pores provide more surface area and expose more sites for attackof aqueous media. The smaller porous filaments within the film yieldgreater dissolution rates, providing a convenient means to controldegradation rates of the porous Si host.

With its high surface area, porous Si is particularly susceptible to airor water oxidation. Once oxidized, nanophase SiO₂ readily dissolves inaqueous media, and surfactants or nucleophiles accelerate the process.Si—O bonds are easy to prepare on porous Si by oxidation, and a varietyof chemical or electrochemical oxidants can be used. Thermal oxidationin air tends to produce a relatively stable oxide, in particular if thereaction is performed at >600° C. Ozone oxidation, usually performed atroom temperature, forms a more hydrated oxide that dissolves quickly inaqueous media.

Slow oxidation of the porous Si surface by dimethyl sulfoxide (DMSO),when coupled with dissolution of the newly formed oxide by HF, is a mildmeans to enlarge the pores in porous Si films. Aqueous solutions ofbases such as KOH can also be used to enlarge the pores after etching.Electrochemical oxidation, in which a porous Si sample is anodized inthe presence of a mineral acid such as H₂SO₄, yields a fairly stableoxide. Oxidation imparts hydrophilicity to the porous structure,enabling the incorporation and adsorption of hydrophilic drugs orbiomolecules within the pores. Aqueous oxidation in the presence ofvarious ions including Ca²⁺ generates a calcified form of porous Si thathas been shown to be bioactive and is of particular interest for in vivoapplications. Calcification can be enhanced by application of a DCelectric current.

The fusogenic liposome-coated nanoparticles of the disclosure provide adevice and method for drug delivery and tissue and disease (e.g., tumor)monitoring. A drug-delivery fusogenic liposome-coated nanoparticlescomposition can include any number of candidate drugs depending upon thetype of condition, tissue, or cancer to be treated. A candidate drug maybe “physically” trapped within the pores of the silicon particle, or,the pores themselves may be chemically modified to bind the candidatedrug. Such a drug can include in the general sense a peptide,polypeptide, small molecule agent, nucleic acid and combinationsthereof.

More specifically, “physical trapping” is similar to building a ship ina bottle, where the “ship” is the candidate drug and the “bottle” is thenanometer-scale pores in the porous Si particle. Small molecules can betrapped in the porous matrix by oxidizing the porous Si around themolecule. Since oxidation of silicon adds two atoms of oxygen per atomof Si to the material, there is a significant increase in volume of thematrix upon oxidation. This has the effect of swelling the pore wallsand shrinking the free volume inside the pores, and under theappropriate conditions, molecules present in the pores during oxidationbecome trapped in the oxide matrix. One embodiment of the trappingprocess is the increased concentration of the active ingredient whichoccurs during the trapping process. The crystals may present anegatively charged environment and an active ingredient, such asproteins and other drugs, may be concentrated in the crystals to levelsmuch higher than the free concentration of the active ingredient insolution. This can result in 10 to 100 fold or more increase in activeingredient concentration when associated with a crystal. The oxidizingcan be performed at repeated intervals by performing layered oxidation.For example, a biological agent or drug can be trapped in the pores bycontrolled addition of oxidants. Oxidation of the freshly prepared(hydride-terminated) porous Si material results in an effectiveshrinking of the pores. This occurs because the silicon oxide formed hasa larger volume than the Si starting material. If a drug is also presentin the solution that contains the oxidant, the drug becomes trapped inthe pores. Furthermore the porous silicon oxide can comprise a higherconcentration of a biological agent or drug than a non-oxidized Sihydride material.

The free volume in a porous Si film is typically between 50 and 80%.Oxidation should reduce this value somewhat, but the free volume isexpected to remain quite high. Most of the current drug deliverymaterials are dense solids and can deliver a small percentage of drug byweight.

Various approaches to load a molecular payload into a porous Si hosthave been explored, and they can be grouped into the following generalcategories: covalent attachment, physical trapping, and adsorption.

Covalent attachment provides a convenient means to link a biomolecularcapture probe to the inner pore walls of porous Si for biosensorapplications, and this approach can also be used to attach drugmolecules, peptides and the like. As described elsewhere herein, linkinga biomolecule via Si—C bonds tends to be a more stable route than usingSi—O bonds due to the susceptibility of the Si—O species to nucleophilicattack.

One of the more common approaches is to graft an organic molecule thatcontains a carboxyl species on the distal end of a terminal alkene. Thealkene end participates in the hydrosilylation reaction, bonding to theSi surface and leaving the carboxy-terminus free for further chemicalmodification. One such linker molecule is undecylenic acid, whichprovides a hydrophobic 10 carbon aliphatic chain to insulate the linkerfrom the porous Si surface. The drug payload can be attached directly tothe carboxy group of the alkene, or it can be further separated from thesurface with a PEG linker. Due to the stability of the Si—C bond,hydrosilylation is good way of attaching a payload to porous Si. Thepayload is only released when the covalent bonds are broken or thesupporting porous Si matrix is degraded.

In yet another embodiment, electrostatic adsorption can be used,essentially an ion exchange mechanism that holds molecules more weakly.Electrostatics is a useful means to affect more rapid drug delivery, asopposed to covalent or physical trapping approaches that release drugover a period of days, weeks, or months.

The affinity of a porous Si particle for a particular molecule can becontrolled with surface chemistry. The surface of oxidized porous Si hasa point of zero charge at a pH of around 2, and so it presents anegatively charged surface to most aqueous solutions of interest. At theappropriate pH, porous SiO₂ spontaneously adsorbs positively chargedproteins such as serum albumin, fibrinogen, protein A, immunoglobulin G(IgG), or horseradish peroxidase, concentrating them in the process.

A calcium silicate porous silicon core is useful for providing a dualrole of condensing the anionic genetic payload with high loadingefficiency, and of emitting photoluminescence to allow for particletracking. The calcium silicate is cationic, which allows for strongelectrostatic interactions with the anionic nucleotides to form a stablecluster of genes and particles. Another aspect of the calcium silicatepSiNPs is the quick dispersion and degradation in intercellularenvironment when the liposomal coating is shed by membrane fusion uponuptake; delayed separation or degradation of the condenser from thenucleotide payloads can result in excretion of the entire cluster, asthe cell recognizes the foreign materials to be inoperable. Data hasdemonstrated that fusogenic particles containing a core calcium silicatepSi quickly degrades and lose photoluminescence without the liposomalprotection in the cell cytoplasm. On the other hand, the non-fusogenicparticles remain intact within endosomes and lysosome, to retain the pSiphotoluminescence. The quick degradation of calcium silicate pSiNPsallows for release of, e.g., siRNA into the cytoplasm to undergo RNAinterference and gene silencing.

Porous Si can also be made hydrophobic, and hydrophobic molecules suchas the steroid dexamethasone or serum albumin can be loaded into thesenanostructures. Hydrophilic molecules can also be loaded into suchmaterials with the aid of the appropriate surfactant. The native hydridesurface of porous Si is hydrophobic. Such techniques have been used forshort-term loading and release. Because water is excluded from thesehydrophobic surfaces, aqueous degradation and leaching reactions tend tobe slow. The grafting of alkanes to the surface by hydrosilylation iscommonly used to prepare materials that are stable in biological media;this stability derives in large part from the ability of the hydrophobicmoieties to locally exclude water or dissolved nucleophiles.

Other drugs (e.g., cargo) or “active ingredient” that can be used withthe porous silicon particles of the disclosure include any one or anycombination of the following, but are not limited to, anti-angiogeniccompounds such as bevacizumab, ranibizumab, pegaptanib, and othercompounds in the angiogenic cascade. Anti-cancer drugs such as, forexample, chemotherapeutic compounds and/or derivatives thereof (e.g.,5-fluorouracil, vincristine, vinblastine, cisplatin, doxyrubicin,adriamycin, tamocifen, etc.). Also included are glucocorticosteroidssuch as dexamethasone, triamcinolone acetonide, fluocinolone acetonideand other comparable compounds in the corticosteroid and cortisenefamilies. Also included are compounds such as antacids,anti-inflammatory substances, coronary dilators, cerebral dilators,peripheral vasodilators, anti-infectives, psychotropics, anti-manics,stimulants, anti-histamines, laxatives, decongestants, vitamins,gastrointestinal sedatives, anti-diarrheal preparations, anti-anginaldrugs, vasodilators, anti-arrhythmics, anti-hypertensive drugs,vasoconstrictors and migraine treatments, anti-coagulants andanti-thrombotic drugs, analgesics, anti-pyretics, hypnotics, sedatives,anti-emetics, anti-nauseants, anti-convulsants, neuromuscular drugs,hyper- and hypoglycemic agents, thyroid and anti-thyroid preparations,diuretics, anti-spasmodics, uterine relaxants, mineral and nutritionaladditives, anti-obesity drugs, anabolic drugs, erythropoietic drugs,anti-asthmatics, bronchodilators, expectorants, cough suppressants,mucolytics, drugs affecting calcification and bone turnover andanti-uricemic drugs. Specific drugs include gastro-intestinal sedativessuch as metoclopramide and propantheline bromide; antacids such asaluminum trisilicate, aluminum hydroxide, ranitidine and cimetidine;anti-inflammatory drugs such as phenylbutazone, indomethacin, naproxen,ibuprofen, flurbiprofen, diclofenac, dexamethasone, prednisone andprednisolone; coronary vasodilator drugs such as glyceryl trinitrate,isosorbide dinitrate and pentaerythritol tetranitrate; peripheral andcerebral vasodilators such as soloctidilum, vincamine, naftidrofuryloxalate, co-dergocrine mesylate, cyclandelate, papaverine and nicotinicacid; anti-infective substances such as erythromycin stearate,cephalexin, nalidixic acid, tetracycline hydrochloride, ampicillin,flucloxacillin sodium, hexamine mandelate and hexamine hippurate;neuroleptic drugs such as flurazepam, diazepam, temazepam,amitryptyline, doxepin, lithium carbonate, lithium sulfate,chlorpromazine, thioridazine, trifluperazine, fluphenazine,piperothiazine, haloperidol, maprotiline hydrochloride, imipramine anddesmethylimipramine; central nervous stimulants such as methylphenidate,ephedrine, epinephrine, isoproterenol, amphetamine sulfate andamphetamine hydrochloride; antihistamic drugs such as diphenhydramine,diphenylpyraline, chlorpheniramine and brompheniramine; anti-diarrhealdrugs such as bisacodyl and magnesium hydroxide; the laxative drug,dioctyl sodium sulfosuccinate; nutritional supplements such as ascorbicacid, alpha tocopherol, thiamine and pyridoxine; anti-spasmodic drugssuch as dicyclomine and diphenoxylate; drugs affecting the rhythm of theheart such as verapamil, nifedipine, diltiazem, procainamide,disopyramide, bretylium tosylate, quinidine sulfate and quinidinegluconate; drugs used in the treatment of hypertension such aspropranolol hydrochloride, guanethidine monosulphate, methyldopa,oxprenolol hydrochloride, captopril and hydralazine; drugs used in thetreatment of migraine such as ergotamine; drugs affecting coagulabilityof blood such as epsilon aminocaproic acid and protamine sulfate;analgesic drugs such as acetylsalicylic acid, acetaminophen, codeinephosphate, codeine sulfate, oxycodone, dihydrocodeine tartrate,oxycodeinone, morphine, heroin, nalbuphine, butorphanol tartrate,pentazocine hydrochloride, cyclazacine, pethidine, buprenorphine,scopolamine and mefenamic acid; anti-epileptic drugs such as phenytoinsodium and sodium valproate; neuromuscular drugs such as dantrolenesodium; substances used in the treatment of diabetes such astolbutamide, disbenase glucagon and insulin; drugs used in the treatmentof thyroid gland dysfunction such as triiodothyronine, thyroxine andpropylthiouracil, diuretic drugs such as furosemide, chlorthalidone,hydrochlorthiazide, spironolactone and triamterene; the uterine relaxantdrug ritodrine; appetite suppressants such as fenfluraminehydrochloride, phentermine and diethylproprion hydrochloride;anti-asthmatic and bronchodilator drugs such as aminophylline,theophylline, salbutamol, orciprenaline sulphate and terbutalinesulphate; expectorant drugs such as guaiphenesin; cough suppressantssuch as dextromethorphan and noscapine; mucolytic drugs such ascarbocisteine; anti-septics such as cetylpyridinium chloride,tyrothricin and chlorhexidine; decongestant drugs such asphenylpropanolamine and pseudoephedrine; hypnotic drugs such asdichloralphenazone and nitrazepam; anti-nauseant drugs such aspromethazine theoclate; haemopoietic drugs such as ferrous sulphate,folic acid and calcium gluconate; uricosuric drugs such assulphinpyrazone, allopurinol and probenecid; and calcification affectingagents such as biphosphonates, e.g., etidronate, pamidronate,alendronate, residronate, teludronate, clodronate and alondronate.

Insofar as the disclosure contemplates including a virtually unlimitednumber of drugs, in vitro pharmacokinetic studies can be used todetermine the appropriate configuration of the porous silicon particlesfor each drug. The drug-conjugated silicon particles can be monitoredonce delivered to a subject. For example, light intensity fromluminescent silicon nanoparticles (LPSiNPs) can be measured using a lowpower spectrophotometer. Using such methods the half-life, delivery andcollection of drugs and/or LPSiNPs can be monitored.

The luminescent spectrum used in particle identification can readily bemeasured with inexpensive and portable instrumentation such as a CCDspectrometer or a diode laser interferometer. Removal of a drug from theLPSiNPs can result in a change in the luminescence of the LPSiNPs as awavelength shift in the spectrum. Such techniques can be used to enablenon-invasive sensing through opaque tissue.

In any of the foregoing embodiments, the silicon particle carrying cargo(e.g., loaded or bound to a drug or agent) or free of any agent and/orwherein the particles are luminescent or non-luminescent areencapsulated in a liposome suitable for fusion with a cell membrane(e.g., fusogenic liposomes). Moreover, these liposomes may be modifiedor unmodified. If modified, the modification can include a targetingmoiety as described further herein.

Fusogenic liposome formulations can be prepared from any number of knownlipids. The liposomal vesicle can be made from a plurality of lipids,wherein the liposomes have a diameter between 10 and 500 nm or anydiameter there between (e.g., 20-400, 50-300, 60-250, 100-200, 120-180,140-160 nm etc.). In a further embodiment, the liposomes are unilamellarliposomes or micelles. In another embodiment, the liposomes aremultilamellar liposomes. In another embodiment, the plurality of lipidscomprise phospholipids selected from phosphatidylcholine, phosphatidicacid, phosphatidylethanolamine, phosphatidylglycerol,phosphatidylserine, lysophosphatidylcholine, and/or any derivativethereof. In yet another embodiment, the phospholipid derivatives areselected from1,2-di-(3,7,11,15-tetramethylhexadecanoyl)-sn-glycero-3-phosphocholine,1,2-didecanoyl-sn-glycero-3-phosphocholine,1,2-dierucoyl-sn-glycero-3-phosphate,1,2-dierucoyl-sn-glycero-3-phosphocholine,1,2-dierucoyl-sn-glycero-3-phosphoethanolamine,1,2-dilinoleoyl-sn-glycero-3-phosphocholine,1,2-dilauroyl-sn-glycero-3-phosphate,1,2-dilauroyl-sn-glycero-3-phosphocholine,1,2-dilauroyl-sn-glycero-3-phosphoethanolamine,1,2-dilauroyl-sn-glycero-3-phospho-(1T-rac-glycerol),1,2-dimyristoyl-sn-glycero-3-phosphate,1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine,1,2-dimyristoyl-sn-glycero-3-phosphoglycerol,1,2-dimyristoyl-sn-glycero-3-phosphoserine,1,2-dioleoyl-sn-glycero-3-phosphate,1,2-dioleoyl-sn-glycero-3-phosphocholine,1,2-dioleoyl-sn-glycero-3-phosphoethanolamine,L-alpha-phosphatidyl-DL-glycerol,1,2-dioleoyl-sn-glycero-3-phosphoserine,1,2-dipalmitoyl-sn-glycero-3-phosphate,1,2-dipalmitoyl-sn-glycero-3-phosphocholine,1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine,1,2-dipalmitoyl-sn-glycero-3-phosphoglycerol,1,2-dipalmitoyl-sn-glycero-3-phosphoserine,1,2-distearoyl-sn-glycero-3-phosphate,1,2-distearoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine,1,2-distearoyl-sn-glycero-3-phosphoglycerol, egg sphingomyelin, egg-PC,hydrogenated Egg PC, hydrogenated Soy PC,1-myristoyl-sn-glycero-3-phosphocholine,1-palmitoyl-sn-glycero-3-phosphocholine,1-stearoyl-sn-glycero-3-phosphocholine,1-myristoyl-2-palmitoyl-sn-glycero 3-phosphocholine,1-myristoyl-2-stearoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-myristoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine,1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol,1-palmitoyl-2-stearoyl-sn-glycero-3-phosphocholine,1-stearoyl-2-myristoyl-sn-glycero-3-phosphocholine,1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, and/or1-stearoyl-2-palmitoyl-sn-glycero-3-phosphocholine. In a furtherembodiment, the liposomes further comprise cholesterol. In yet anotherembodiment, the liposomes further comprise polyethylene glycol. Forexample, in one embodiment, the fusogenic liposomal formulation can beprepared from 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] (DSPE-PEG), and 1,2-dioleoyl-3-trimethylammonium-propane(DOTAP) in molar ratio of 76.2:3.8:20. For experimental controls,non-fusogenic formulations can be synthesized in the same manner, but atmolar ratio of 80:0:20 (i.e., lacking PEG). DSPE-PEG (methoxy) may bereplaced in the same molar ratio with carboxylic acid—(DSPE-PEG(carboxy)) or maleimide—(DSPE-PEG (maleimide)) functionalized PEG lipidfor further surface modifications such as with targeting moieties. Forexample, use of DSPE-PEG (carboxy) or DSPE-PEG (meleimide) are usefulfor modification with antibodies or targeting peptides, respectively. Inanother embodiment, the liposomes further comprise one or moresite-targeting moieties. Examples of site-targeting moieties include,but are not limited to, peptides, aptamers, antibodies, and antibodyfragments (e.g., F(ab′)₂, Fab, and scFv).

The term “lipid” refers to any suitable material resulting in a bilayersuch that a hydrophobic portion of the lipid material orients toward thebilayer while a hydrophilic portion orients toward the aqueous phase.Amphipathic lipids are used as the primary lipid vesicle structuralelement. Hydrophilic characteristics derive from the presence ofphosphato, carboxylic, sulfato, amino, sulfhydryl, nitro, and other likegroups. Hydrophobicity can be conferred by the inclusion of groups thatinclude, but are not limited to, long chain saturated and unsaturatedaliphatic hydrocarbon groups and such groups substituted by one or morearomatic, cycloaliphatic or heterocyclic group(s). Typical amphipathiccompounds are phosphoglycerides and sphingolipids, representativeexamples of which include phosphatidylcholine, phosphatidylethanolamine,phosphatidylserine, phosphatidylinositol, phosphatidic acid,palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine,lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine,dioleoylphosphatidylcholine, distearoylphosphatidylcholine ordilinoleoylphosphatidylcholine could be used. Other compounds lacking inphosphorus, such as sphingolipid and glycosphingolipid families are alsowithin the group designated as lipid. Additionally, the amphipathiclipids described above may be mixed with other lipids includingtriglycerides and sterols.

The term “neutral lipid” refers to any of a number of lipid specieswhich exist either in an uncharged or neutral zwitterionic form atphysiological pH. Such lipids include, for examplediacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide,sphingomyelin, cephalin, and cerebrosides.

The term “non-cationic lipid” refers to any neutral lipid as describedabove as well as anionic lipids. Examples of anionic lipids includecardiolipin, diacylphosphatidylserine and diacylphosphatidic acid.

The term “cationic lipid” refers to any of a number of lipid specieswhich carry a net positive charge at physiological pH. Such lipidsinclude, but are not limited to, DODAC, DOTMA, DDAB, DOTAP, DC-Chol andDMRIE. Additionally, a number of commercial preparations of cationiclipids are available which can be used in the disclosure. These carriersare improved by the addition of PEG-modified lipids and, in particular,PEG-modified ceramide lipids. The addition of PEG-modified lipidsprevents particle aggregation and provides a means for increasingcirculation lifetime and increasing the delivery of the lipid-nucleicacid particles to the target cells. Moreover, it has been found thatcationic lipids fuse more readily with the target cells and, thus, theaddition of neutrally charged PEG-modified ceramide lipids does not maskor diminish the positive charge of the carrier liposomes.

Exemplary lipids useful for formulation of liposome include, but are notlimited to:

1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethyleneglycol)-2000] (DSPE-PEG2000 (carboxy)).

Generally, lipids dissolved in chloroform are mixed and solvent isevaporated to produce a dry film. In an embodiment where hydrophobicpayload is embedded in the liposomal membrane, the hydrophobic payloadsolution is mixed with the lipid solution to be dried into the filmtogether. The payload-entrapped porous silicon-based core is used tohydrate the fusogenic liposome film with gentle pipetting. The mixturewill turn cloudy to indicate liposome formation. In some embodiments,the mixture can be heated at about 40° C. for 10 min before undergoingmechanical extrusion, e.g., 20 times through polycarbonate membrane with200 nm pores. Excess payload is washed out by centrifugation three timeswith deionized water. FIG. 1B-D illustrates a schematic of the liposomalporous silicon nanoparticle synthesis. FIG. 1E depicts a schematicrepresentation of a fusogenic liposomal porous silicon micro- and/ornanoparticle composition of the disclosure.

Typically the liposomal coating is comprised of pro-fusogenic lipids andmoieties. For example, DMPC can acts as the structural backbone of theliposome, with a relatively low phase transition temperature (T_(m)=24°C.). The low transition temperature gives the L_(α) liquid crystal phaseat room temperature and body temperature. The L_(α) phase is the morefluidic, dynamic, and permeable structure that allows for the wide sizerange (100-400 nm) of extruded liposomal coatings and an easier fusionpotential. DOTAP is a cationic lipid useful for the electrostaticattraction toward the anionic plasma membrane. PEGylated lipids was alsouseful in fusion; though the exact mechanistic role of PEG is not yetknown, it is hypothesized that PEG electrostatically binds watermolecules to dehydrate the lipid head groups, which leads to structuralasymmetry in the lipid alignment and drives double-leaflet tosingle-leaflet fusion as the energetically favorable route, similar tohow SNARE proteins anchor and pull vesicles into merging with plasmamembranes endogenously; in fact, neuronal SNAREs have been observed topromote PEG-mediated fusion.

It is further contemplated herein, that the liposomal layerencapsulating the silicon particle can be adapted for site-targeting, bytethering targeting moieties (peptides, aptamers, antibodies, antibodyfragments, sugar or glycolipids) on or in the liposomal layer, which canguide the silicon particle and its cargo selectively to desired sites,thereby facilitating local drug delivery and therapeutic effects. Forexample, various reactive groups can be employed to tether targetinggroups to the lipids making up the liposomes disclosed herein, such assulfhydryl-reactive groups, maleimides, haloacetyls, pyridyldisulfides,thiosulfonates, and vinylsulfones; carboxyl-to-amine reactive groups,such as carbodiimides (e.g., EDC); amine-reactive groups, such as NHSesters, imidoesters, pentafluorophenyl esters, hydroxylmethyl phosphine;aldehyde-reactive groups, such as hydrazides, and alkoxyamines;photoreactive groups, such as diazinine, and aryl azide; and hydroxyl(nonaqueous)-reactive groups, such as isocyanates.

The targeting moieties bound to the surface of the liposome may varyfrom small haptens of from about 125-200 dalton molecular weight to muchlarger antigens with molecular weights of at least about 6 kD, butgenerally of less than 10⁶ kD. Proteinaceous ligand and receptors are ofparticular interest. Since agents/cargo incorporated in the siliconparticles contained in the liposome may be indiscriminate with respectto cell type in its action, a targeted delivery system offers asignificant improvement over randomly injecting non-specific liposomes.

A number of procedures can be used to covalently attach eitherpolyclonal or monoclonal antibodies to a liposome bilayer.Antibody-targeted liposomes can include monoclonal or polyclonalantibodies or fragments thereof such as scFV, Fab, or F(ab′)₂, so longas they bind efficiently to the antigenic epitope on the target cells.

As mentioned above, the disclosure also provides embodiments wherein thefusogenic liposome-coated nanoparticles comprise a targetingmoiety/molecule. A targeting moiety/molecule can include, but is notlimited to, a ligand or an antibody (including antibody fragments) thatspecifically binds to its corresponding target, for example, a receptoron a cell surface or an antigen. Thus, for example, where the targetingmolecule is an antibody or fragment thereof, the fusogenicliposome-coated nanoparticles will specifically bind (target) cells andtissues bearing the epitope to which the antibody or antibody fragmentis directed. Thus, a targeting molecule refers generally to allmolecules capable of reacting with or otherwise recognizing or bindingto a receptor or polypeptide on a target cell (e.g., it's cognate pair).Any known ligand or targeting molecule can be used. Examples oftargeting peptides that can be manipulated and cloned or linked toproduce a fusogenic liposome-coated nanoparticles are ample in theliterature. In general, any peptide ligand can be used or fragmentsthereof based on the receptor-binding sequence of the ligand. Inimmunology, such a peptide domain is referred to as an epitope, and theterm epitope may be used herein to refer to a ligand recognized by areceptor. For example, a ligand comprises the sequence of a protein orpeptide that is recognized by a binding partner on the surface of atarget cell, which for the sake of convenience is termed a receptor.However, it should be understood that for purposes of the disclosure,the term “receptor” encompasses signal-transducing receptors (e.g.,receptors for hormones, steroids, cytokines, insulin, and other growthfactors), recognition molecules (e.g., MHC molecules, B- or T-cellreceptors), nutrient uptake receptors (such as transferrin receptor),lectins, ion channels, adhesion molecules, extracellular matrix bindingproteins, and the like that are located and accessible at the surface ofthe target cell.

Various cell-types can be targeted. For example, antigen presentingcells (APCs), including leukocytes, may be targeted by making fusogenicliposome-coated nanoparticles comprising targeting molecules thatrecognize targets on the APCs. In operation, the fusogenicliposome-coated nanoparticles bind the targets fuse with the membrane,are internalized by the cells, and release the nanoparticle's contentsinto the cell. Examples of receptors that can be targeted or used astargeting moieties include, for example, the following receptors, orreceptors for: E-selectin, CD3, CD 4, CD8, CD11, CD 14, CD 34, CD 123,CD 45Ra, CD64, E-cadherin, ICAM-1, interleukins, interferons, tumornecrosis factors, E-cadherin, Fc, MCH, CD 36 and other integrins,chemokines, Macrophage Mannose receptor and other lectin receptors, B7,CD's 40, 50, 80, 86 and other costimulatory molecules, Dec-205,scavenger receptors and toll receptors, see also Guermonprez et al.(Annu. Rev. Immunol., 2002).

The various embodiments provided herein are generally directed tosystems and methods for producing a drug delivery device that candeliver cargo for treating or diagnosis of various diseases or disordersincluding viral and bacterial infections, cancers, tumors and other cellproliferative diseases and disorders, inflammatory diseases anddisorders and tissue damage. In addition, the disclosure providesimmunization techniques that boost drug delivery or promote drug actionor improve immunogen processing associate with a silicon nanoparticle ofthe disclosure. Such methodology can include activating dendritic cellsand other inflammatory cells and stimulating an immune response.

The composition comprising the liposome encapsulating the silicon nano-and/or micro-particle, can be formulated for enteral delivery,parenteral delivery, topical delivery, or by inhalation. Theliposome-containing silicon particle of the disclosure can be formulatedfor in vitro and in vivo administration using techniques known in theart.

A pharmaceutical composition of the disclosure is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical),transmucosal, and rectal administration. Solutions or suspensions usedfor parenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy to administer by a syringe. It must be stable under theconditions of manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it is useful to include isotonic agents, for example, sugars,polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating theliposomal silicon particle composition, e.g. a composition disclosedherein, in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

In a particular embodiment, one or more liposomal formulations of thedisclosure are prepared with carriers that will protect the compoundagainst rapid elimination from the body, such as a controlled releaseformulation, including use of polyethylene glycol, implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations should be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc.

Data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (e.g., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. The methods andcompositions of the disclosure are applicable to a wide range ofspecies, e.g., humans, non-human primates, horses, cattle, pigs, sheep,goats, dogs, cats, rabbits, guinea pigs, hamsters, rats, and mice.

The liposomal silicon particle compositions and formulations disclosedherein, including pharmaceutical compositions comprising saidformulations, can be used to treat any number of diseases or disorderthat require delivery of a peptide, small molecule, nucleic acid (e.g.,siRNA) into a cell.

The working examples below are provided to illustrate, not limit, theinvention. Various parameters of the scientific methods employed inthese examples are described in detail below and provide guidance forpracticing the invention in general.

EXAMPLES

Fusogenic Silicon Nanoparticle Synthesis.

Fusogenic liposome films were made with a lipid solution in a mix ofDMPC:DSPE-PEG:DOTAP at molar ratio of 76.2:3.8:20, along with theaddition of 21 μL of 1.25 mg/ml of(2Z)-2-[(E)-3-(3,3-dimethyl-1-octadecylindol-1-ium-2-yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole(DiI).

Porous silicon nanoparticles (pSiNPs) were prepared by electrochemicaletching of single-crystal silicon wafers in an ethanolic aqueous HFsolution followed by removal of the porous layer and ultrasonicfracture.

The liposomal mixture was dried into a film, and hydrated with theporous silicon or calcium silicate-deposited porous silicon nanoparticlesolution. The hydrated suspension was heated to 40° C. under magneticstirring for 20 min, and mechanically extruded twenty times throughpolycarbonate membrane with 200 nm pores.

FIG. 1B-H shows the particle synthesis schematic and characterizationdata. Scattering and microscopic data of the fusogenic liposome-coatedpSi (F-pSi) confirm hydrodynamic diameter of approximately 190 nm. FIG.1E-F show the particle characterization by transmission electronmicroscopy (TEM).

Drug Loading into Porous Silicon Particles.

siRNA payloads were loaded within the pSi core cluster through calciumsilicate sealing chemistry to approximately 20 wt % loading efficiency.Other oligonucleotide-loaded nanoplatforms, such as lipid-basednanoparticles and mesoporous silica-polymer hybrid systems, have anaverage loading efficient of 1-14 wt % (Table 1). In particular,comparably sized 200 nm particles can only load less than 5 wt % inthese materials. Thus, the fusogenic porous silicon particles of thedisclosure demonstrated a four-fold increase in oligonucleotide-loadingefficiency, which can in turn enhance cellular gene knockdownefficiency.

TABLE 1 siRNA loading efficiency by wt. % comparison between FusogenicpSiNPs and conventional platforms. siRNA Loading Particle Size (wt. %)Fusogenic liposome-coated pSiNP 200 nm 20-25%     Lipid-basedNanoparticles 50-200 nm 1-14% ⁴¹⁻⁵¹ Mesoporous Silica-Polymer Hybrid NPs60-200 nm 1-10% ⁵²⁻⁵⁵

In Vitro Uptake Behavior of Fusogenic Particles

Neuro2a cells were incubated with fusogenic liposomes and non-fusogenicliposomes for 1 h, and were visualized under confocal microscopy.Fusogenic particles transferred lipophilic DiI from the liposomalmembrane to the plasma membrane to stain the cell outline, whereasnon-fusogenic liposomes were found to localize in distinct groups in thecell cytoplasm, which is characteristic of endosomal or lysosomalcompartmentalization.

Particles were loaded with hydrophilic (calcein) or lipophilic (DiI)dyes to evaluate differences in dye localization, and thus, particleuptake (FIG. 4). Calcein was chosen as the model-siRNA payload, as it isan anionic dye which typically cannot penetrate through the cellmembrane, very similar to siRNA behavior. On the other hand, DiI wasused as a way to confirm fusogenic uptake, as DiI is a lipophilic dyethat is loaded into the liposomal bilayer. If the particle fuses uponuptake, the DiI would transfer and spread from the liposomal bilayerinto the plasma membrane bilayer.

F-pSi loaded with DiI in the liposome membrane successfully transferredthe DiI into the cell membrane and dispersed the calcein signalthroughout the cell cytoplasm, indicating uptake via membrane fusion. Inaddition, minimal photoluminescence signal from the pSiNPs wasdetected—attributed to rapid degradation without liposomal protectionpost-fusion. On the other hand, non-fusogenic liposomal pSi (NF-pSi)loaded with DiI or calcein were localized dense clusters within the cellcytoplasm, indicating endocytosis. Moreover, calcein signalsco-localized with pSi photoluminescence signals in concentrated spots.The pSi signal indicates that the core remains intact with the calceindye in endosomes/lysosomes due to liposomal protection. Since membranefusion did not take place, we hypothesize that the liposome-coated pSiparticle is endocytosed.

Using LysoTracker Red, the lysosomal intracellular compartments werestained as shown in FIGS. 4(b) and (e). The fusogenic and non-fusogenicparticles were loaded with calcein and applied to cells. Results showedthat while fusogenic particles showed dispersed calcein signals that didnot co-localized with lysosomes, non-fusogenic particles showed calceinco-localization with lysosomal compartments. Further, DiI-loadedfusogenic and non-fusogenic particles conjugated withmacrophage-targeting peptide (MTP) were prepared and treated tomacrophages. The MTPs were also tagged with a 6-FAM dye to allow forfluorescence monitoring. Fusogenic particles were shown to fuse alongspecific parts of the cell membrane where MTP-FAM signals alsoco-localized, indicating that the MTP-FAM successfully anchors theparticles to specific macrophage membrane receptors to allow forlocalized fusion. It is also notable that MTPs seemed to expedite thefusion process by quickly anchoring the particles to cell membranesurface, as only half the incubation time was needed to achievecomparable level of fusion. Non-fusogenic particles conjugated withMTP-FAM demonstrated the same localization as in the same particleswithout MTP. The MTP-FAM and DiI signals co-localized in clusters withinthe cytoplasm, indicative of endosomal and lysosomalcompartmentalization.

Cell Impermeable Dye-Loaded Particles for Cytosolic Staining

Calcein is an anionic cell impermeable fluorescent dye. Due to itsstrong negative charge, it can be entrapped in porous siliconnanoparticles using the same calcium chloride interaction as nucleicacid payloads. Porous silicon particles were placed in an aqueoussolution containing the Calcein and 1M or greater concentration of CaCl₂solution. The solution was mixed and purified by centrifugation,resulting in Calcein-loaded calcium silicate coated porous siliconnanoparticles, or pSiNPs (Ca-pSi-Calcein). Unloaded Calcein was washedout by centrifugation three times in deionized water.

HeLa cells were incubated with fusogenic liposome-coated particles andnon-fusogenic liposome-coated particles, and were visualized underconfocal microscopy over the following time intervals: 30 min, 1 h, 2 h,4 h, and 8 h. Fusogenic liposome-coated particles were observed torelease Calcein directly into the cell cytoplasm, and disperse themthroughout the cell. The lack of red luminescence signal from poroussilicon indicates its quick dissolution in the intracellular environmentdue to loss of shielding from liposome coating from the fusogenic uptake(FIG. 6). In contrast, non-fusogenic liposome-coated particles wereco-localized with the calcein signals, in concentrated spots in thecytoplasm. The observation indicates that the non-fusogenicliposome-coated particles were endocytosed into the cells as a whole,and the maintenance of liposomal coating around the porous silicon-basedcore prevented core dissolution and loss of luminescence (FIG. 7).

In Vivo Gene Knockdown and Therapeutic Effect

Infected mice were tested with varying formulations to test for fusionand IRF5 knockdown effectiveness as anti-bacterial therapeutics. 6-weekold female Balb/C mice were intratracheally infected with Staph. aureusbacteria 24 h prior to intravenous tail-vein injection of saline (PBS),targeted non-fusogenic particles loaded with therapeutic siRNA(NF-siIRF5-MTP), targeted fusogenic particles loaded with controlnon-therapeutic siRNA (F-siLuc-MTP), or targeted fusogenic particlesloaded with therapeutic siRNA (F-siIRF5-MTP). The mice were observed for7 days following treatment for survival, and tallied to obtain theresults shown in FIG. 8A.

FIG. 8B shows the average days of survival from each treatment group,and the statistical significance. From single way ANOVA and post hoccomparisons using Tukey's HSD test, the data show that mice injectedwith F-siIRF5-MTP particles had significantly higher average number ofsurvival days than all other formulations, PBS, NF-siIRF5-MTP, andF-siLuc-MTP (p level<0.05, [F (3, 20)=8.78, p=0.001].

In all treatment groups, mice surviving past 4 days showed normalhealthy behavior and no visible outward signs of distress or illness.

Fusogenic Silicon Particle Stability

The particle stabilities in phosphate-buffered saline (PBS) was observedfor 28 days by measuring hydrodynamic diameter changes by DLS (FIG. 9).Fusogenic liposome-coated particles (F-CapSi) were extruded to aninitial average hydrodynamic diameter of approximately 140 nm.RVG-conjugation to the maleimide-terminated PEG (RVG-F-CapSi) extendedthe size to approximately 180 nm. The size remained highly stable forthe first 7 days, before increasing gradually over the next 3 weeks.

Antibody-Conjugated Particles

Antibodies can be conjugated to the surface of the liposome by a similarPEG interaction used with targeting peptide conjugations. A fusogenicliposome film was made of mix of DMPC:DSPE-PEG (carboxy):DOTAP at molarratio of 76.2:3.8:20. The mixture was dried into a film, and hydratedwith porous silicon or metal (Ca) silicate-deposited porous siliconnanoparticle solution. The hydrated suspension was heated to 40° C.under magnetic stirring for 20 min, and mechanically extruded twentytimes through polycarbonate membrane with 200 nm pores. The amine groupon the lysine residue of the Fc region of the antibody was conjugated tothe carboxyl-terminated PEG to form an amide bond via1-ethyl-3-(3-dimethylaminopropyl)carbodiimide/sulfo-N-hydroxysuccinimide(EDC/sulfo-NHS) chemistry.

In order to verify cellular uptake by membrane fusion, lipophilic(2Z)-2-[(E)-3-(3,3-dimethyl-1-octadecylindol-1-ium-2-yl)prop-2-enylidene]-3,3-dimethyl-1-octadecylindole(DiI) fluorescent dye, was loaded into the liposomal membrane. Particleswere treated to Neuro2a cells, and incubated for 1 h for cellularuptake. Fusogenic particles were able to successfully transfer thelipophilic DiI from the liposomal membrane to the plasma membrane,whereas particles coated with non-fusogenic liposomes were found tolocalize in distinct groups in the cell cytoplasm, which ischaracteristic of endosomal/lysosomal uptake (FIG. 12, left panel).

Next, the difference between fusogenic and non-fusogenic liposomalcoating in intracellular localization of hydrophilic payloads wereinvestigated. Calcein, a highly anionic cell-impermeable dye was loadedwithin the porous silicon core using calcium chloride entrapment, andencapsulated in fusogenic and non-fusogenic liposomes. FIG. 10 shows theabsorbance and fluorescence spectra of Calcein loaded in liposome-coatedparticles. Liposomal encapsulation of Calcein-loaded particles allowedde-quenching of the fluorescence emission.

The photoluminescence of calcium silicate-coated particles was alsostudied; the calcium silicate/silicon oxide shell or magnesiumsilicate/silicon oxide shell displays a strong ability to passivate thesurface of the silicon nanostructures, yielding increased intrinsicphotoluminescence from the material. FIG. 11 shows photoluminescencespectra obtained at different times during the course of the reactionbetween pSiNPs and CaCl2 (or MgCl2) solution. During the reaction, theintensity of photoluminescence gradually increased, attributed to thepassivation of nano-radiative carrier traps on the pSiNP surface.Additionally, the peak wavelength of photoluminescence showed apronounced blue shift as the reaction progressed. Both these phenomena(increase in photoluminescence intensity and blue shift of thephotoluminescence spectrum) are indicative of the growth of apassivating surface layer on the silicon nanocrystallites. The observedblue shift is typical of a quantum confined nanoparticle, whose emissionwavelength is strongly dependent on size and expected to blue shift asthe quantum confined silicon domains become smaller. Because it isintimately tied to the host silicon matrix, the intrinsicphotoluminescence of the nanoconstruct can be used to monitordegradation of the matrix and, by inference, the release of payload inan in vitro or in vivo experiment.

The particles were treated to HeLa cells and visualized under confocalmicroscopy over time. The right panel on FIG. 12 shows the cellularuptake of the particles after 8 h of incubation. Fusogenicliposome-coated particles were observed to release the Calcein payloadinto the cell cytoplasm, and disperse them throughout the cell. The lackof luminescence signal from calcium silicate-coated porous siliconindicates its quick dissolution in the intracellular environment due tolack of shielding from fused liposome. In contrast, non-fusogenicliposome-coated particles were co-localized with the calcein signals, inconcentrated spots in the cytoplasm. The observation indicates that thenon-fusogenic liposome-coated particles were endocytosed into the cellsas a whole, and the maintenance of liposomal coating around the poroussilicon-based core prevented core dissolution.

Targeting Peptide-Conjugated Particles

Rabies virus glycoprotein (RVG, (CCGG)YTIWMPEN PRPGTPCDIFTNSRGKRASNG(SEQ ID NO:1)) is a Neuro2a mouse neuroblast-targeting peptide, which isconjugated to the maleimide-terminated PEG of the liposomal coat viamaleimide-thiol/cysteine covalent binding interaction. To observeinteractions with cells under confocal microscopy, the RVG peptide wastagged with 5-FAM dye, and the liposomal membrane with lipophilic DiI.Targeting-peptide conjugation did not seem to affect fusogenicity of theliposome coating, and were only observed to accelerate fusion and uptakerate compared to the fusogenic liposome-coated particles withouttargeting peptide.

siRNA-Loaded Particles for Gene Knockdown

Small interfering RNA (siRNA) cargo was loaded into the porous siliconcore by mixing the porous silicon particles with siRNA dissolved indeionized water and 3M CaCl₂ and washing by centrifugation.

For siRNA knockdown test in Neuro2a cells, peptidylprolyl isomerase B(PPIB/siPPIB) was chosen as the target gene (FIG. 14). The siPPIB-loadedF-pSi demonstrated knockdown efficiency of approximately 95% at 100 nM.By contrast, the common transfection agent lipofectamine achieved only90% knockdown efficiency under similar conditions. The non-fusogeniccontrol nanoparticles NF-pSi demonstrated only 30% knockdown 100 nM,with high variability.

In Vivo Macrophage-Targeting and Infection Homing

Balb/C mice were intratracheally infected with Staph. aureus to inducelung infection. A macrophage/monocyte-targeting peptide was attached tothe F/NF-pSi loaded with calcein and DiI, and the formulations wereadministered into Staph. aureus infected mice via intravenous injection,to observe for targeting efficacy to macrophages and the macrophagehoming to the infected lungs (FIG. 15). Harvested lungs of mice injectedwith calcein-loaded particles were homogenized for FACS quantificationof calcein accumulation in infected lungs, as shown in FIGS. 15(a-c).Lungs of mice injected with DiI-loaded particles were fixed forfluorescence histological evaluation, as shown in FIGS. 15(d-k). Despitetargeting peptide attachment, NF-pSi showed no visible homing to lungsin both Calcein-loaded FACs data and the DiI-loaded fluorescenthistological evaluation. F-pSi without targeting peptide showed minimalaccumulation in both assessments, whereas F-pSi with targeting peptidedemonstrated clear targeting to macrophages and subsequent homing toinfected lungs in both FACS and fluorescent histology. Thus, themacrophage-targeting peptide is necessary in in vivo homing ofnanoparticles to macrophages.

With the MTP targeting peptide conjugation as described herein, thenanoparticles loaded with either Calcein or DiI were able tosuccessfully home to the infected lung, whereas particles without theMTP peptide and the non-fusogenic particles could not. Healthy lungsalso showed no visible homing, as there are fewer macrophages that arerecruited. With the validation that the MTP peptide homing in infectedlungs, formulations loaded with therapeutic siIRF5 was tested fortherapeutic efficacy.

The disclosure demonstrates that fusogenic liposome-coated pSi systemare able to bypass endocytosis to achieve greater gene knockdownefficacy compared to non-fusogenic formulations. Furthermore, in vivotargeting to macrophages/monocytes was successfully demonstrated, andthe payload-delivered macrophage/monocytes homed effectively to infectedlungs. With M1 phenotype-suppressive siRNA delivery to the macrophages,that data show that the fusogenic system can achieve improved andeffective immunogenic clearance of infection potentially due to higherphagocytic activity, inflammation mitigation, and tissue-healing.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

What is claimed is:
 1. A fusogenic liposome-coated porous siliconnanoparticle comprising: (a) a porous silicon nanoparticle corematerial, (b) a plurality of cargo molecules that are physicallyassociated with the porous silicon nanoparticle core material, (c) ametal silicate on a surface of the porous silicon nanoparticle corematerial, and (d) fusogenic liposome coating around the porous siliconnanoparticle core material, the plurality of cargo molecules, and themetal silicate.
 2. The fusogenic liposome-coated porous siliconnanoparticle of claim 1, wherein said fusogenic liposome coatingcomprises 1,2-dimyristoyl-sn-glycero-3-phosphocholine,1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000], and 1,2-dioleoyl-3-trimethylammonium-propane.
 3. Thefusogenic liposome-coated porous silicon nanoparticle of claim 1,wherein the porous silicon nanoparticle core material comprises oxidizedsilicon.
 4. The fusogenic liposome-coated porous silicon nanoparticle ofclaim 1, wherein the metal silicate comprises a calcium or magnesiumsilicate.
 5. The fusogenic liposome-coated porous silicon nanoparticleof claim 4, wherein the metal silicate forms a shell on an outer surfaceof the porous silicon nanoparticle core material.
 6. The fusogenicliposome-coated porous silicon nanoparticle of claim 5, wherein thereare no gaps between the porous silicon nanoparticle core material andthe calcium or magnesium silicate shell.
 7. The fusogenicliposome-coated porous silicon nanoparticle of claim 1, wherein theplurality of cargo molecules are located within a plurality of pores inthe porous silicon nanoparticles core material.
 8. The fusogenicliposome-coated porous silicon nanoparticle of claim 1, wherein theporous silicon nanoparticle core material is chemically oxidized, andwherein the plurality of cargo molecules are physically trapped within aplurality of pores in the porous silicon nanoparticle core material. 9.The fusogenic liposome-coated porous silicon nanoparticle of claim 1,wherein the plurality of pores contain a nucleic acid.
 10. The fusogenicliposome-coated porous silicon nanoparticle of claim 1, wherein thefusogenic liposome coating comprises a mixture of DMPC, DOTAP, andDSPE-PEG (methoxy).
 11. The fusogenic liposome-coated porous siliconnanoparticle of claim 1, wherein the fusogenic liposome coatingcomprises a mixture of DMPC, DOTAP, and DSPE-PEG (carboxy).
 12. Thefusogenic liposome-coated porous silicon nanoparticle of claim 1,wherein the fusogenic liposome coating comprises a mixture of DMPC,DOTAP, and DSPE-PEG (maleimide).
 13. The fusogenic liposome-coatedporous silicon nanoparticle of claim 1, wherein the porous siliconnanoparticle core material has a hydrodynamic diameter ranging fromabout 10-100 nm.
 14. The fusogenic liposome-coated porous siliconnanoparticle of claim 1, wherein the fusogenic liposome has ahydrodynamic diameter ranging from about 100-400 nm.
 15. The fusogenicliposome-coated porous silicon nanoparticle of claim 1, wherein atargeting molecule is conjugated to the fusogenic liposome coating. 16.The fusogenic liposome-coated porous silicon nanoparticle of claim 1,wherein an antibody is conjugated to the fusogenic liposome coating. 17.The fusogenic liposome-coated porous silicon nanoparticle of claim 1,wherein a hydrophilic cargo molecule is physically trapped within aplurality of pores in the porous silicon nanoparticle-core material. 18.The fusogenic liposome-coated porous silicon nanoparticle of claim 1,wherein a nucleic acid cargo molecule is physically trapped within aplurality of pores in the porous silicon nanoparticle-core material. 19.The fusogenic liposome-coated porous silicon nanoparticle of claim 1,wherein a small molecule cargo molecule is physically trapped within aplurality of pores in the porous silicon nanoparticle core material. 20.A method of delivering a nucleic acid payload to a cell comprisingcontacting the cell with the fusogenic liposome-coated porous siliconnanoparticle of claim
 18. 21. The fusogenic liposome-coated poroussilicon nanoparticle of claim 1, wherein the porous silicon nanoparticlecore material is prepared by electrochemical or chemical etching ofcrystalline silicon.